Like other enveloped viruses, paramyxoviruses spread infections from cell to cell and from host to host in the form of particles which are formed by budding from infected cell membranes. Paramyxovirus matrix (M) proteins organize the assembly process, linking together the viral glycoproteins and the viral ribonucleoproteins (vRNPs). Interactions between M proteins and nucleocapsid (NP) proteins are the driving force for active incorporation of vRNPs into virions, and hence are fundamentally important for the infectivity of nsRNA virus particles. Here, we propose to investigate paramyxovirus M protein interactions that drive the packaging of vRNPs into particles. Our first aim is to manipulate and inhibit paramyxovirus genome packaging interactions by targeting the nucleocapsid proteins. We will attempt to achieve competitive inhibition of M-NP interactions using foreign proteins and peptides that incorporate into budding particles using the same interactions that normally direct vRNPs into virions. We also propose a series of experiments to define parameters that govern M-NP interactions among the paramyxoviruses. Our second aim is to define and manipulate paramyxovirus genome packaging interactions by targeting the matrix proteins. Guided by second-site mutations and the recently-determined M protein structure, we have defined PIV5 M mutations that enhance interactions with NP. Consequences of enhanced M-NP interaction will be assessed both in transfected cells using mini-genome assays and in the context of recombinant virus. We hypothesize that M-NP interaction strength in wt virus is carefully balanced. M-NP interaction that is too strong will benefit virus assembly, but will also lead to inappropriate generation of M- bound vRNPs during the early phases of infection, thereby impairing viral transcription and/or genome replication. We will also define individual roles for two distinct clusters of second-site mutations on the M protein surface, with the hypothesis that the C-terminal surface indirectly influences virus assembly and NP binding through interactions with host factors.